Our principal accomplishments and on-going projects during the last year have been: 1) What specifies the correct timing of these guidance and differentiation signals to form the conserved and stereotyped branching patterns of the neuro-vascular network? During angiogenesis, a primary capillary network undergoes intensive vascular remodeling and develops into a hierarchical vascular branching network. Our previous studies have shown that peripheral sensory nerves provide a unique anatomical template that determines the arterial branching pattern in the developing skin. This alignment facilitates access to oxygen and nutrients for the nerves. At the molecular level, two distinct mechanisms underlie the congruence of sensory nerve and arterial vessel branching: nerve-derived VEGF-A controlling arterial differentiation, and nerve-derived Cxcl12 controlling vessel branching and alignment with nerves (Li et al. Dev Cell, 2013). We are interested in whether oxygen-starved sensory nerves may trigger Cxcl12 and VEGF-A expression that govern the precise pattern of arterial branching along with the nerves. The fact that the regulatory regions of Cxcl12 and VEGF-A genes contain a HIF-dependent hypoxia-responsive enhancer encourages us to carry out a nerve-specific knockout of the Hif-1&#945; or Hif-2&#945; subunit or both. One phenotype we expect to observe in these mutants would be defective expression of Cxcl12 and VEGF-A in nerves resulting in defective nerve-vessel alignment and arterial differentiation. 2) How does the neuro-vascular network influence organ physiology, regeneration, and diseases? We are engaged in a new project for studying the role of the neuro-vascular association during tissue repair or in disease conditions. Whole-mount immunofluorescence microscopy has revealed that adult ear skin maintains the neuro-vascular bundle, suggesting that the association reflects the mutual requirement of nerve and vessel in the function and maintenance of both networks. Using this adult ear skin vasculature model, we are currently studying peripheral nerve regeneration and re-vascularization in the ear skin regeneration/wound healing. 3) How does neuronal axon guidance molecules influence vascular branching network formation? Several molecules with attractive and repulsive properties have been found to modulate the proper guidance of both networks. We focus our studies on understanding the role of Neuropilin-2 (NRP2) which regulates neuronal axon guidance as a co-receptor for class 3 Semaphorins (SEMA3s)/PlexinAs signaling. In the lymphatic vascular network formation, SEMA3G and SEMA3F negatively regulate VEGF-C-mediated lymphatic endothelial cell proliferation and sprouting through interaction with the NRP2/PlexinA receptor complex (Uchida et al. Biol Open, 2015). Given a mutually exclusive expression of NRP2 (veins) and SEMA3G (arteries) in the blood vasculature, we have found that SEMA3G/NRP2 complex-mediated signaling influences artery-vein communication in the developing skin. Combined, our data suggest that SEMA3/NRP2 signaling functions as a negative regulator to form an intricate vascular and lymphatic network. In a similar line of research, we have collaborative studies on the role of multiple signaling pathways on sprouting lymphangiogenesis in the skin, stemming from our previous discovery that TGF signaling controls lymphatic vessel development (James et al. Development, 2013)